猪链球菌荧光定量PCR检测方法的建立及临床应用

Establishment of Fluorescence Quantitative PCR for Streptococcus suis Detection and Its Clinical Application

为快速诊断和检测猪链球菌病,根据GenBank中已登录的猪链球菌抗原基因保守区域序列,设计并合成特异性引物及Taqman探针,通过优化荧光定量PCR反应体系及扩增条件,构建了快速、准确检测猪链球菌的Taqman荧光定量PCR检测方法(Taqman FQ-PCR),测定了该方法的灵敏性、重复性和特异性,并对疑似猪链球菌临床感染样本进行了检测。结果显示:所建立的Taqman FQ-PCR方法检测灵敏度为1×10~2拷贝/μL;对3种不同浓度的猪链球菌重组质粒进行3次重复扩增,变异系数均小于3%,说明重复性好;特异性检测显示,只有猪链球菌样本的扩增曲线呈阳性反应,其他致病菌均无荧光信号;采用此方法,对30份临床疑似猪链球菌感染样本进行检测,发现有27份样本为阳性,比采用国标法检测得到的结果更加灵敏。结果表明,建立的FQPCR方法具有灵敏性高、重复性好、特异性强等特点,可用于猪链球菌的快速诊断和检测。

In order to diagnose and detect swine streptococcosis rapidly,the specific primers and Taqman probes were designed according to the conservative regional sequence of the antigen gene of Streptococcus suis（SS） in GenBank. By optimizing the reaction system and amplification conditions of real-time PCR,Taqman real-time PCR method was established,which could detect the SS quickly and accurately. The sensitivity,repeatability and specificity of this method were tested,and suspected clinical infection samples were also detected. The results showed that the sensitivity of the established Taqman FQ-PCR method was 1×10^2 copy/μL. Three recombinant plasmids with different concentrations of SS were amplified 3 times,and the variation coefficient was less than 3%,which showed good repeatability. The specific test showed that only the amplification curve of SS was positive and the other pathogens had no fluorescent signal. Thirty samples of suspected SS infection were detected by this method,and 27 samples were tested positive,which was more sensitive than the national standard. In conclusion,the FQ-PCR method was sensitive,reproducible and specific,which was beneficial to the rapid diagnosis and detection of SS.